Because of the importance of calcium as an intracellular messenger and regulator, a wide variety of techniques have been developed for measuring intracellular free calcium concentrations [Ca.sup.2+ ].sub.i (see reference 4). The most successful of these techniques uses dyes or proteins which change absorption or luminescence upon binding Ca.sup.2+ ions. Currently the most popular of these methods is to monitor the fluorescence of a BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetracetic acid) like indicator dye compound called "quin2" (see references 4-7). When this method is used to measure intracellular free calcium in mammalian cells, quin2 is loaded into intact cells by incubating them with a membrane-permeant ester derivative. Cytosolic esterases then split off the ester groups, leaving the membrane-impermeant quin2 tetra-anion trapped in the cytosol. The fluorescence of quin2 provides a measure of cytosolic free calcium concentrations. It is calibrated by interpolation between the limiting fluorescence levels obtained by forcing the trapped dye to known saturatingly high as well as vanishingly low levels of calcium.
Although quin2 has revealed much important biological information, it has severe and acknowledged limitations (see references 5 and 7). For example, quin2's preferred excitation wavelength of 339 nm is too short since it falls within the ultraviolet range. UV irradiation excites significant autofluorescence from the cells being monitored. In addition, such UV irradiation could cause biological side effects in the cells being tested. Also, it is known that light in this UV range does not penetrate microscope optics very well.
A second problem with quin2 is the fact that its extinction coefficient (&lt;5000) and fluorescence quantum yield (0.03 to 0.14), though comparable to dansyl groups in aqueous solution, are too low. These spectroscopic properties mean that quin2 loadings of several tenths of millimolar or more are necessary to overcome cell autofluorescence. In some cell types, this much loading significantly buffers [Ca.sup.2+ ].sub.i transients.
A third problem with quin2 is the fact that it signals Ca.sup.2+ by increasing its fluorescence intensity without much shift in either excitation or emission wavelengths. Unfortunately, fluorescence intensity is also dependent on other poorly quantified or variable factors such as illumination intensity, emission collection efficiency, dye concentration, and effective cell thickness in the optical beam. As a result, it would be better to have an indicator dye that responded to calcium by shifting wavelengths while still maintaining strong fluorescence. The ratio of the fluorescences of two suitably chosen wavelengths would then signal calcium while cancelling out most or all of the possible variability due to instrument efficiency and content of effective dye.
A fourth problem with quin2 is that it does not measure calcium at micromolar levels. The high effective affinity of quin2 for Ca.sup.2+ is ideal for measuring levels near or below typical resting values of [Ca.sup.2+ ].sub.i, i.e., near 10.sup.-7 M. Unfortunately, at micro levels of calcium or above, quin2 approaches saturation so that further changes in fluorescence intensity are small. As a result, dyes with weaker affinities are needed to quantify calcium at these elevated levels.
Finally, the selectivity of quin2 for calcium over magnesium and heavy metal divalent cations could bear improvement. Quin2 binds Mg.sup.2+ with a dissociation constant of 1-2 mM. Though Mg.sup.2+ has little effect on the fluorescence of quin2 when excited at 339 nm, variations in [Mg.sup.2+ ].sub.i would affect the effective affinity for Ca.sup.2+ and the calibration scale for the fluorescence signals. Also, cells with unusually high levels of exchangeable heavy metals can give falsely low readings of [Ca.sup.2+ ].sub.i because the heavy metals quench quin2 fluorescence (see reference 8). Conversely, dye loading could perturb [Mg.sup.2+ ].sub.i or chelate heavy metals that are important in cell functions.
Thus there is a need for a new class of fluorescent indicator dyes that are specific for calcium ions. Such dyes are needed to measure calcium concentrations in aqueous solutions, especially biological fluids and even more especially, calcium concentrations within living cells. To allow measurement of calcium concentrations on an absolute scale, the new fluorescent indicator compounds should exhibit a wavelength shift upon Ca.sup.2+ -binding. The new compounds should also overcome some of the specific problems associated with use of the known Ca-specific indicator, quin2. More specifically, as compared to quin 2, the new compounds should show much stronger fluorescence, a somewhat weaker affinity for Ca.sup.2+ and a better selectivity against magnesium and heavy metals.